A sensing device applied to an analyte molecule of a liquid sample and a buffer flow has at least one first inlet, at least one second inlet, a micro-flow channel, and at least one immobilization element. The first inlet is for inputting the liquid sample. The second inlet is for inputting the buffer flow. The micro-flow channel communicates with the first inlet and the second inlet. The immobilization element is for immobilizing sensing molecules for the analyte molecules. The analyte sample flow and the buffer flow, in the reverse direction, in the micro-flow channel enable the association and disassociation kinetics to be obtained. The present invention further provides a sensing system and a sensing method using the sensing device.